PKD2L2 Antibody

What is PKD2L2 and how does it relate to the polycystin protein family?

PKD2L2 (Polycystic Kidney Disease 2-Like 2) is a member of the polycystin protein family that functions as a transient receptor potential cation channel . It shares structural similarities with PKD2, which is implicated in autosomal-dominant polycystic kidney disease. Unlike PKD2, which interacts with proteins like Hax-1 and is linked to the actin cytoskeleton, PKD2L2 displays distinct interaction patterns and potentially different functional roles . Understanding these differences is critical when designing experiments targeting specific members of the polycystin family.

What applications are PKD2L2 antibodies validated for?

PKD2L2 antibodies are validated for multiple experimental applications including:

• Western Blotting (WB)

• Immunofluorescence (IF)

• Immunohistochemistry (IHC)

• Enzyme-Linked Immunosorbent Assay (ELISA)

Researchers should verify the specific validation for their antibody of interest, as validation can vary between commercial sources. For instance, some antibodies like those from Proteintech (13872-1-AP) are specifically validated for ELISA applications with reactivity to human and mouse samples .

How do you determine the specificity of a PKD2L2 antibody?

Determining antibody specificity requires multiple validation approaches. For PKD2L2 antibodies, researchers should:

• Review manufacturer validation data showing the antibody recognizes endogenous levels of PKD2L2 protein

• Confirm cross-reactivity profiles (e.g., whether the antibody cross-reacts with other polycystin family members)

• Perform control experiments using knockout/knockdown cells

• Include positive and negative control tissues with known PKD2L2 expression patterns

For comparison, the PKD2 antibody (D1A7) demonstrates high specificity as it recognizes endogenous levels of total PKD2 protein without cross-reacting with PKD1 or PKD3 . Similar validation should be sought for PKD2L2 antibodies.

What are the optimal sample preparation protocols for PKD2L2 antibody applications?

Sample preparation protocols differ by application:

For Western Blotting:

• Use a complete protein extraction buffer containing protease inhibitors

• Optimal dilution ratios typically range from 1:500 to 1:1000

• Expected molecular weight for PKD2L2 is approximately 74 kDa

For Immunofluorescence:

• Fixation with 4% paraformaldehyde preserves PKD2L2 subcellular localization

• Permeabilization with 0.1-0.5% Triton X-100

• Recommended dilution ranges from 1:100 to 1:200

• Include appropriate counterstains to visualize cellular structures

The storage buffer and conditions significantly impact antibody performance. Most PKD2L2 antibodies should be stored in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3, and kept at -20°C for optimal stability .

How can researchers distinguish between PKD2L2 and closely related proteins in experimental systems?

Distinguishing PKD2L2 from related proteins requires:

• Selection of highly specific antibodies with validated non-cross-reactivity to other family members

• Comparative analysis with other polycystin antibodies

• Using protein-specific domains as controls

Studies have demonstrated that while PKD2 and PKD2L (a PKD2-related protein) share high sequence homology, they display distinct interaction patterns. For example, PKD2 interacts with Hax-1, but PKD2L does not, despite their structural similarities . Researchers can leverage these differential interactions as specificity controls.

Additionally, co-immunoprecipitation experiments can confirm specific protein-protein interactions characteristic of PKD2L2 versus other family members, similar to approaches used to validate PKD2-Hax-1 interactions in mammalian cells .

What are the most effective co-localization approaches for studying PKD2L2 interactions with other proteins?

For effective co-localization studies of PKD2L2:

• Double immunofluorescence staining with:

  • PKD2L2 antibody

  • Organelle-specific markers

  • Potential interacting proteins

• Advanced microscopy techniques:

  • Confocal microscopy for subcellular co-localization

  • Super-resolution microscopy for nanoscale interaction analysis

  • Live-cell imaging for dynamic interaction studies

• Controls and validation:

  • Use of pre-absorption controls

  • Sequential antibody application to avoid cross-reactivity

  • Quantitative co-localization analysis with statistical validation

Drawing from studies of PKD2, which demonstrated co-localization with Hax-1 in the endoplasmic reticulum and cellular processes like lamellipodia , researchers can design similar approaches for PKD2L2 to identify its subcellular localization and potential interaction partners.

How can researchers optimize co-immunoprecipitation protocols for studying PKD2L2 protein interactions?

Optimizing co-immunoprecipitation (co-IP) for PKD2L2 requires:

• Cell model selection:

  • Stably transfected cell lines expressing tagged PKD2L2

  • Cells with endogenous PKD2L2 expression

• Lysis conditions:

  • Non-denaturing buffers to preserve protein-protein interactions

  • Appropriate detergent selection (NP-40 or Triton X-100 at 0.5-1%)

• Antibody considerations:

  • Pre-clearing lysates to reduce non-specific binding

  • Using both N-terminal and C-terminal targeting antibodies

  • Including appropriate negative controls (IgG from the same species)

• Validation approaches:

  • Reciprocal co-IP experiments

  • Mass spectrometry analysis of co-precipitated proteins

This approach mirrors successful co-IP experiments used to confirm PKD2-Hax-1 interactions in stably transfected HeLa cells expressing HA-tagged human PKD2 and GST-Hax-1 fusion proteins .

What are common sources of variability in PKD2L2 antibody performance across applications?

Common sources of variability include:

• Antibody-specific factors:

  • Lot-to-lot variations in commercial antibodies

  • Polyclonal versus monoclonal differences in epitope recognition

  • Storage conditions affecting antibody stability

• Sample-related factors:

  • Protein denaturation affecting epitope accessibility

  • Cross-reactivity with homologous proteins

  • Post-translational modifications masking epitopes

• Protocol-specific considerations:

  • Fixation methods altering protein conformation

  • Buffer compositions affecting antibody binding kinetics

  • Blocking reagents causing background issues

To minimize variability, researchers should:

• Perform validation with positive control samples of known PKD2L2 expression

• Use recombinant antibodies when available for superior lot-to-lot consistency

• Document detailed protocols and antibody lot information for reproducibility

How should researchers address contradictory results between different PKD2L2 antibodies?

When encountering contradictory results:

• Systematic validation approach:

  • Test multiple antibodies targeting different epitopes of PKD2L2

  • Compare polyclonal versus monoclonal antibody results

  • Verify antibody specificity using knockout/knockdown controls

• Technical considerations:

  • Evaluate whether the contradictions are application-specific

  • Assess if antibody epitopes might be differentially accessible in various applications

  • Consider whether post-translational modifications affect epitope recognition

• Resolution strategies:

  • Use orthogonal detection methods (mass spectrometry, RNA-level validation)

  • Employ tagged recombinant proteins for interaction studies

  • Document antibody performance systematically across different applications

How are PKD2L2 antibodies being used to understand its role in disease mechanisms?

Current research applications include:

• Subcellular localization studies:

  • Determining whether PKD2L2, like PKD2, localizes primarily to the endoplasmic reticulum

  • Investigating potential translocation under pathological conditions

• Protein interaction networks:

  • Identifying PKD2L2-specific binding partners to understand functional roles

  • Comparing PKD2L2 interactions with those of other polycystin family members

• Expression pattern analysis:

  • Tissue-specific expression profiling in normal versus disease states

  • Correlation of expression levels with disease progression

• Functional studies:

  • Investigating cation channel activity in different cellular contexts

  • Evaluating roles in cellular signaling pathways

These approaches build upon methodologies established for PKD2, which has been demonstrated to interact with the cytoskeleton through Hax-1 and cortactin , potentially revealing distinct functions for PKD2L2.

What are the latest methodological advances in PKD2L2 antibody applications for structural biology?

Recent methodological advances include:

• Proximity labeling approaches:

  • BioID or APEX2 fusion proteins to identify proximal interacting partners

  • Complementation assays to confirm direct interactions

• Advanced imaging techniques:

  • Live-cell super-resolution microscopy for dynamic PKD2L2 localization

  • Correlative light and electron microscopy for ultrastructural context

• Structural biology applications:

  • Using antibodies as crystallization chaperones

  • Single-particle cryo-EM studies of PKD2L2 channel complexes

  • Hydrogen-deuterium exchange mass spectrometry with antibody epitope mapping

• Cell-free protein synthesis systems:

  • Production of functional PKD2L2 for structural and functional studies

  • Incorporation into artificial membranes for channel activity measurement

These approaches leverage both traditional antibody applications and novel technologies to advance understanding of PKD2L2 structure-function relationships.

How do PKD2L2 antibodies compare with antibodies against other polycystin family members in research applications?

When selecting between polycystin family antibodies, researchers should consider:

• The specific research question and required application

• Validated reactivity for the target species

• Cross-reactivity profiles with other family members

• Whether conformational or linear epitopes are preferred

Understanding these differences ensures appropriate antibody selection and experimental design for polycystin family research.

What experimental conditions differentiate optimal PKD2L2 antibody performance from other polycystin antibodies?

Optimal performance conditions vary across polycystin family antibodies:

• Fixation preferences:

  • PKD2L2 antibodies may perform differently with various fixation methods

  • Comparison with PKD2 antibodies, which work well with paraformaldehyde fixation for ER localization studies

• Extraction conditions:

  • Different detergent requirements for membrane protein solubilization

  • Buffer composition optimization for each family member

• Epitope accessibility:

  • Consideration of protein topology and membrane insertion

  • Domain-specific antibodies for targeting accessible regions

• Application-specific optimization:

  • Western blotting: Transfer conditions for different molecular weights

  • Immunoprecipitation: Binding kinetics and affinity differences

  • Immunofluorescence: Permeabilization requirements

Researchers should conduct systematic optimization for each antibody rather than assuming protocols can be transferred between family members, even with high sequence homology.